• BMB Reports
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• 제52권8호
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• Yonsei Medical Journal
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• 제59권9호
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• pp.1096-1106
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• 2018

Purpose: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. Materials and Methods: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma ($PPAR-{\gamma}$) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. $PPAR-{\gamma}$ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and $NF-{\kappa}B$ activity was determined by a Caspase 3 Activity Assay Kit or $NF-{\kappa}B$ p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and $PPAR-{\gamma}$ 3'UTR. Results: MiR-128 expression was upregulated and $PPAR-{\gamma}$ expression was downregulated in plasma from AD patients and $amyloid-{\beta}$ $(A{\beta})-treated$ primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased $A{\beta}-mediated$ cytotoxicity through inactivation of $NF-{\kappa}B$ in MCN and N2a cells. Moreover, $PPAR-{\gamma}$ was a target of miR-128. $PPAR-{\gamma}$ upregulation attenuated $A{\beta}-mediated$ cytotoxicity by inactivating $NF-{\kappa}B$ in MCN and N2a cells. Furthermore, $PPAR-{\gamma}$ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and $NF-{\kappa}B$ activity in MCN and N2a cells. Conclusion: MiR-128 inhibitor decreased $A{\beta}-mediated$ cytotoxicity by upregulating $PPAR-{\gamma}$ via inactivation of $NF-{\kappa}B$ in MCN and N2a cells, providing a new potential target in AD treatment.

• Biomolecules & Therapeutics
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• 제29권5호
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• pp.551-561
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• 2021

Thyroid cancer is the most common endocrine malignancy. Patients with well-differentiated thyroid cancers, such as papillary and follicular cancers, have a favorable prognosis. However, poorly differentiated thyroid cancers, such as medullary, squamous and anaplastic advanced thyroid cancers, are very aggressive and insensitive to radioiodine treatment. Thus, novel therapies that attenuate metastasis are urgently needed. We found that both PDGFC and PDGFRA are predominantly expressed in thyroid cancers and that the survival rate is significantly lower in patients with high PDGFRA expression. This finding indicates the important role of PDGF/PDGFR signaling in thyroid cancer development. Next, we established a SW579 squamous thyroid cancer cell line with 95.6% PDGFRA gene insertion and deletions (indels) through CRISPR/Cas9. Protein and invasion analysis showed a dramatic loss in EMT marker expression and metastatic ability. Furthermore, xenograft tumors derived from PDGFRA geneedited SW579 cells exhibited a minor decrease in tumor growth. However, distant lung metastasis was completely abolished upon PDGFRA gene editing, implying that PDGFRA could be an effective target to inhibit distant metastasis in advanced thyroid cancers. To translate this finding to the clinic, we used the most relevant multikinase inhibitor, imatinib, to inhibit PDGFRA signaling. The results showed that imatinib significantly suppressed cell growth, induced cell cycle arrest and cell death in SW579 cells. Our developed noninvasive apoptosis detection sensor (NIADS) indicated that imatinib induced cell apoptosis through caspase-3 activation. In conclusion, we believe that developing a specific and selective targeted therapy for PDGFRA would effectively suppress PDGFRA-mediated cancer aggressiveness in advanced thyroid cancers.

• Journal of Ginseng Research
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• 제46권6호
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• pp.738-749
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• 2022

Background: Ginseng possesses antitumor effects, and ginsenosides are considered to be one of its main active chemical components. Ginsenosides can further be hydrolyzed to generate secondary saponins, and 20(R)-panaxotriol is an important sapogenin of ginsenosides. We aimed to synthesize a new ginsengenin derivative from 20(R)-panaxotriol and investigate its antitumor activity in vivo and in vitro. Methods: Here, 20(R)-panaxotriol was selected as a precursor and was modified into its derivatives. The new products were characterized by ¹H-NMR, ¹³C-NMR and HR-MS and evaluated by molecular docking, MTT, luciferase reporter assay, western blotting, immunofluorescent staining, colony formation assay, EdU labeling and immunofluorescence, apoptosis assay, cells migration assay, transwell assay and in vivo antitumor activity assay. Results: The derivative with the best antitumor activity was identified as 6,12-dihydroxy-4,4,8,10,14-pentamethyl-17-(2,6,6-trimethyltetrahydro-2H-pyran-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl(tert-butoxycarbonyl)glycinate (A11). The focus of this research was on the antitumor activity of the derivatives. The efficacy of the derivative A11 (IC₅₀ < 0.3 µM) was more than 100 times higher than that of 20(R)- panaxotriol (IC₅₀ > 30 µM). In addition, A11 inhibited the protein expression and nuclear accumulation of the hypoxia-inducible factor HIF-1α in HeLa cells under hypoxic conditions in a dose-dependent manner. Moreover, A11 dose-dependently inhibited the proliferation, migration, and invasion of HeLa cells, while promoting their apoptosis. Notably, the inhibition by A11 was more significant than that by 20(R)-panaxotriol (p < 0.01) in vivo. Conclusion: To our knowledge, this is the first study to report the production of derivative A11 from 20(R)-panaxotriol and its superior antitumor activity compared to its precursor. Moreover, derivative A11 can be used to further study and develop novel antitumor drugs.

• Advances in nano research
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• 제12권6호
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• pp.639-652
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• 2022

The prolyl 3-hydroxylase family member 4 (P3H4), is associated with post-translational modification of fibrillar collagens and aberrantly activated in cancer leading to tumor progression. However, its role in clear cell renal cell carcinoma (ccRCC) is still unknown. Here we reported that P3H4 was highly expressed in renal cancer tissues and significantly positive correlated with poor prognosis. Knockdown of P3H4 inhibited the proliferation, migration and metastasis of renal cancer cells in vitro and in vivo, and also, overexpression of it enhanced the oncogenic process. Mechanistically, P3H4 depletion decreased the levels of GDF15-MMP9 axis and repressed its downstream signaling. Further functional studies revealed that inhibition of GDF15 suppressed renal cancer cell growth and GDF15 recombinant human protein (rhGDF15) supplementation effectively rescued the inhibitory effect induced by P3H4 knockdown. Moreover, decreased levels of MMP9 caused by inhibition of P3H4-GDF15 signaling constrained the expression of PD-L1 and suppression of P3H4 accordingly promoted anti-tumor immunity via stimulating the infiltration of CD4+ and CD8+ T cells in syngeneic mice model. Taken together, our findings firstly demonstrated that P3H4 promotes ccRCC progression by activating GDF15-MMP9-PD-L1 axis and targeting P3H4-GDF15-MMP9 signaling pathway can be a novel strategy of controlling ccRCC malignancy.

• Journal of Life Science
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• 제32권2호
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• pp.148-154
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• 2022

Focal adhesion kinase (FAK) is known to regulate cell adhesion, migration, and mechanotransduction in focal adhesions (FAs). However, studies on how FAK activity is regulated in the plasma membrane microdomains according to the composition of extracellular matrix (ECM) proteins are still lacking. A genetically encoded fluorescence resonance energy transfer (FRET)-based biosensor can provide useful information on the activity of intracellular signals with high spatiotemporal resolution. In this study, we analyzed the FAK activities in lipid raft (detergent-resistant membrane) and non-lipid raft (non-detergent-resistant membrane) microdomains using FRET-based membrane targeting FAK biosensors (FAK-Lyn and FAK-KRas biosensors) under four different ECM protein compositions: glass, type 1 collagen, fibronectin, and laminin. Interestingly, FAK activity in response to laminin in a lipid raft microdomain was lower than that in other ECM conditions. Cells subjected to fibronectin showed higher FAK activity in a lipid raft microdomain than that in a non-lipid raft microdomain. Therefore, this study demonstrates that the FAK activity can be distinctively regulated according to the ECM type and the environment of the plasma membrane microdomains.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.73-81
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• 2023

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.

• Journal of Life Science
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• 제33권8호
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• pp.651-662
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• 2023

Peroxisomes, known as microbodies, are a class of morphologically similar subcellular organelles commonly found in most eukaryotic cells. They are 0.2~1.8 ㎛ in diameter and are bound by a single membrane. The matrix is usually finely granular, but occasionally crystalline or fibrillary inclusions are observed. They characteristically contain hydrogen peroxide (H₂O₂) generating oxidases and contain the enzyme catalase, thus confining the metabolism of the poisonous H₂O₂ within these organelles. Therefore, the eukaryotic organelles are greatly dynamic both in morphology and metabolism. Plant peroxisomes, in particular, are associated with numerous metabolic processes, including β-oxidation, the glyoxylate cycle and photorespiration. Furthermore, plant peroxisomes are involved in development, along with responses to stresses such as the synthesis of important phytohormones of auxins, salicylic acid and jasmonic acids. In the past few decades substantial progress has been made in the study of peroxisome biogenesis in eukaryotic organisms, mainly in animals and yeasts. Advancement of sophisticated techniques in molecular biology and widening of the range of genomic applications have led to the identification of most peroxisomal genes and proteins (peroxins, PEXs). Furthermore, recent applications of proteome study have produced fundamental information on biogenesis in plant peroxisomes, together with improving our understanding of peroxisomal protein targeting, regulation, and degradation. Nonetheless, despite this progress in peroxisome development, much remains to be explained about how peroxisomes originate from the endoplasmic reticulum (ER), then assemble and divide. Peroxisomes perform dynamic roles in many phases of plant development, and in this review, we focus on the latest progress in furthering our understanding of plant peroxisome functions, biogenesis, and dynamics.

• Proceedings of the Plant Resources Society of Korea Conference
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• 한국자원식물학회 2023년도 임시총회 및 춘계학술대회
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• pp.50-50
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• 2023

Prostaglandin E2(PGE2), a major product of cyclooxygenase-2 (COX-2), plays an important role in the carcinogenesis of many solid tumors, including colorectal cancer. Because PGE2 functions by signaling through PGE2 receptors (Eps), which regulate tumor cell growth, invasion, and migration, there has been a growing amount of interest in the therapeutic potential of targeting Eps. In the present study, we investigated the role of EP4 on the effectiveness of cordycepin in inhibititing the migration and invasion of HCT116 human colorectal carcinoma cells. Our data indicate that cordycepin suppressed lipopolysaccharide (LPS)-enhanced cell migration and invasion through the inactivation of matrix metalloproteinases (MMP)-9 as well as the down-regulation of COX-2 expression and PGE2 production. These events were shown to be associated with the inactivation of EP4 and activation of AMP-activated protein kinase (AMPK). Moreover, the AMPK inhibitor, compound C, as well as AMPK knockdown via siRNA, attenuated the cordycepin-induced inhibition of EP4 expression. Cordycepin treatment also reduced the activation of CREB. These findings indicate that cordycepin suppresses the migration and invasion of HCT116 cells. Through modulating EP4 expression and the AMPK-CREB signaling pathway. Therefore, cordycepin has the potential to serve as a potent anti-cancer agent in therapeutic strategies against colorectal cancer metastasis.

• BMB Reports
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• 제57권6호
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• pp.281-286
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• 2024

Adult hippocampal neurogenesis plays a pivotal role in maintaining cognitive brain function. However, this process diminishes with age, particularly in patients with neurodegenerative disorders. While small, non-coding microRNAs (miRNAs) are crucial for hippocampal neural stem (HCN) cell maintenance, their involvement in neurodegenerative disorders remains unclear. This study aimed to elucidate the mechanisms through which miRNAs regulate HCN cell death and their potential involvement in neurodegenerative disorders. We performed a comprehensive microarray-based analysis to investigate changes in miRNA expression in insulin-deprived HCN cells as an in vitro model for cognitive impairment. miR-150-3p, miR-323-5p, and miR-370-3p, which increased significantly over time following insulin withdrawal, induced pronounced mitochondrial fission and dysfunction, ultimately leading to HCN cell death. These miRNAs collectively targeted the mitochondrial fusion protein OPA1, with miR-150-3p also targeting MFN2. Data-driven analyses of the hippocampi and brains of human subjects revealed significant reductions in OPA1 and MFN2 in patients with Alzheimer's disease (AD). Our results indicate that miR-150-3p, miR-323-5p, and miR-370-3p contribute to deficits in hippocampal neurogenesis by modulating mitochondrial dynamics. Our findings provide novel insight into the intricate connections between miRNA and mitochondrial dynamics, shedding light on their potential involvement in conditions characterized by deficits in hippocampal neurogenesis, such as AD.